CD40/CD40 ligand interactions have been demonstrated to be critical co-stimulatory molecules in the generation of a successful immune response. We hypothesized that it may be possible to augment the immunostimulatory effects of CD40 stimulation by combining it with cytokine therapy. IL2 has been demonstrated to augment both adaptive and innate immune responses. Thus far, cytokine therapy has concentrated on using combinations of cytokines. We reasoned that the use of CD40 stimulation to promote dendritic cell development and function coupled with the properties of IL2 to promote T cell function would result in synergistic anti-tumor effects in vivo. Using a highly metastatic murine renal cancer model we found that strong synergistic anti-tumor effects resulted if CD40 stimulation was applied with IL2 therapy whereas no effect was observed using either agent alone. We now propose to delineate the mechanism underlying these anti-tumor effects and optimize the immunotherapeutic potential of this regimen. Toward this goal we have developed 5 specific aims: Specific Aim 1 will assess the effects of CD40 stimulation using agonist antibodies to CD40 in combination with IL2 on various metastatic renal tumor models (using both CD40+ and CD40- tumor lines) to determine the extent at which this approach is efficacious and to determine if specific immunity results upon rechallenge. Specific Aim 2 will explore the immunomodulatory effects of this regimen through the assessment of various components of both the innate (dendritic, NK) and adaptive (T and B) immune system as well as the role of cytokines. Specific Aim 3 will determine the role of CD40 on various tissues in response to this regimen through the use of CD40 -/- mice and various chimeras constructed by them. Specific Aim 4 will ascertain the efficacy of a recombinant soluble ligand for CD40 (srCD40L) to determine if similar immunomodulatory and anti-tumor effects can be obtained with this combination with IL2, particularly in light of recent data demonstrating extreme toxicities associated with antibodies to CD40 given after cytoreductive conditioning. Specific Aim 5 will then examine the effects of CD40 stimulation and IL2 in models in which cytoreductive conditioning is applied to advanced tumor-bearing mice. Both potential toxicities (which will be sought to be overcome), effects on immune reconstitution, and anti-tumor effects will be evaluated. This last model will most closely resemble advanced tumor bearing patients undergoing a debulking procedure followed with immunotherapy to remove the minimal residual disease. This proposal examining induction of costimulation with CD40 followed with IL2, should yield significant insights, not only to the efficacy of this combination approach in cancer, but also as an immunostimulatory regimen for infectious disease.